Download Celecoxib: A Specific COX-2 Inhibitor With Anticancer Properties

Epidemiological data and
preclinical studies have
generated compelling
interest in the potential
use of COX-2 inhibitors
in chemoprevention
and chemotherapy
of human tumors.
Michele Sassi. La Digue Island, c. 2001. Seychelles Islands, Indian Ocean. Photograph.
Celecoxib: A Specific COX-2 Inhibitor With
Anticancer Properties
Alane T. Koki, PhD, and Jaime L. Masferrer, PhD
I
n addition to the well-established pathophysiological role that COX-2 plays in inflammation, recent
evidence implies that this isoform may also be involved in multiple biologic events throughout the tumorigenic
process. Many epidemiological studies demonstrate that nonsteroidal anti-inflammatory drugs (NSAIDs)
reduce the risk of a wide range of tumors. Further, COX-2 is chronically overexpressed in many premalignant,
malignant, and metastatic human cancers, and levels of overexpression have been shown to significantly
correlate to invasiveness, prognosis, and survival in some cancers. Pharmacological studies consistently
demonstrate that COX-2 inhibitors dose-dependently inhibit tumor growth and metastasis in various relevant
animal models of cancer. Importantly, several investigators have also shown COX-2 inhibitors may act
additively or synergistically with currently used cytotoxics and molecularly targeted agents. Here we present
a broad overview of the growing evidence that COX-2 plays a pivotal role throughout oncogenesis and
summarize the rationale to explore the use of COX-2 inhibitors for the prevention and/or treatment of cancer
as a single agent or in combination with current anticancer modalities.
Introduction
The cyclooxygenases are responsible for the conversion of arachidonic acid to prostaglandins (PGs),
and their metabolites play a pivotal role in multiple
physiologic and pathophysiologic processes. Cyclooxygenase-1 (COX-1) is constitutively expressed in
From the Pharmacia Corp, Chesterfield, Missouri.
Address reprint requests to Alane T. Koki, PhD, Pharmacia Corporation, 700 Chesterfield Village Parkway North, Mailzone BB3K,
Chesterfield, MO 63017. E-mail: [email protected]
28 Cancer Control
most tissues and is responsible for maintaining physiologic processes such as gastric and renal protection
and platelet function. In contrast, cyclooxygenase-2
(COX-2) is induced in response to growth factors1,2 (ie,
endothelial growth factor [EGF], vascular endothelial
growth factor [VEGF], fibroblast growth factor [FGF2]), cytokines (eg, tumor necrosis factor-α [TNF-α],
interleukin-α [IL-α], and interleukin-1β [IL-1β]), and
tumor promoters (eg, v-src, v-Ha-ras, HER-2/neu, and
Wnt).3,4 COX-2 is expressed in macrophages, synoviocytes, and endothelial cells in response to inflammation and cellular activation.5-7 Conventional NSAIDs
March/April 2002, Vol. 9, No.2 Supplement
inhibit both COX-1 and COX-2, hence they also disrupt
COX-1 dependent homeostatic functions. Therefore,
molecular-based targeting strategies were employed to
develop specific COX-2 inhibitors to circumvent the
gastric and renal toxicities caused by mixed COX
inhibitors.8-10
In addition to the well-studied role of COX-2 in
acute inflammatory processes, recent work clearly
suggests COX-2-derived metabolites contribute at
multiple points throughout tumorigenesis, including
premalignant hyperproliferation, transformation, maintenance of tumor viability, growth, invasion, and
metastatic spread. Here we summarize the collective
evidence that supports the potential anticancer activity
of COX-2 inhibitors.
Epidemiological Evidence:
COX-2 and Cancer
Epidemiological studies provided the first evidence that COX may be involved in the pathogenesis
of cancer. Several reports indicate NSAIDs can
prevent the development of various human tumors,
including colon,11-18 breast,19-22 lung,19 gastric,23,24 and
esophageal24,25 neoplasias. For example, in a prospective study evaluating data from 12,668 subjects over
12.4 years, the incidence of several cancers was lower
in regular users of aspirin. The incidence rate for all
cancers combined was 0.83 (95% CI, 0.74-0.93), lung
cancer 0.68 (95% CI, 0.49-0.94), breast cancer in
women 0.70 (95% CI, 0.50-0.96), and colorectal cancer
in younger men 0.35 (95% CI, 0.17-0.73).19
These data collectively provide the rationale for
additional clinical trials to stringently evaluate NSAID
efficacy in other types of cancers as well.
COX-2 Expression in Human Tumors
To understand the potential importance and role
of COX-2 in human cancers, we developed and utilized
immunohistochemical techniques to stringently characterize COX-2 in progressing stages of tumorigenesis
Head and neck
Bladder
Cholangiocarcinoma
Hepatocellular
Renal Cell
Breast
Mesothelioma
Endometrial
Pancreatic
Prostate
Basal Cell
Gastric
1A
1B
Fig 1. — COX-2 is up-regulated in various solid tumors. Fig 1a shows up-regulation in head and neck, bladder, renal cell, breast, pancreatic, and prostate
cancer. Fig 1b shows up-regulation in cholangiocarcinoma, hepatocellular (HCC), mesothelioma, endometrial, basal cell, and gastric cancers. Formalinfixed, paraffin-embedded human cancers from archival tissues were prepared by immunohistology using standard methods and stained for COX-2 with
isoform-specific antisera. The red stain depicts the presence of COX-2.
March/April 2002, Vol. 9, No.2 Supplement
Cancer Control 29
in human epithelial and solid tumors. COX-2 was consistently overexpressed in premalignant lesions such as
oral leukoplakia, actinic keratosis, prostatic intraepithelial neoplasia, and carcinoma in situ of the bladder and
breast.11,22,26 COX-2 was also up-regulated in head and
neck, bladder, lung, hepatocellular, pancreatic, mesothelioma, cholangiocarcinoma, gastric, cutaneous, Kaposi’s
sarcoma, and cervical cancers (Fig 1).
In general, COX-2 is up-regulated throughout the
tumorigenic process, from early hyperplasia to metastatic disease (Fig 2). Elevated levels of COX-2 immunoreactivity were primarily detected in the neoplastic
epithelium, inflammatory cells, and vasculature within
and adjacent to tumor nests (Fig 3).
Normal
Ductal CA
DCIS CA
Infiltrating CA
COX-2 was also consistently and more intensely
observed in metastatic lesions compared with the corresponding primary tumor. In general, COX-2 is
expressed in 40% to 80% of neoplastic cells in human
cancers and the extent and intensity of expression is
greater in cancerous than in noncancer cells. Moreover, well- and moderately-differentiated cancers have
significantly higher COX-2 expression than poorly differentiated cancers. COX-2 is also detected in noncancerous cells immediately adjacent to tumor cells
(<2 mm) and in the angiogenic vasculature within
tumors and in pre-existing blood vessels adjacent to
tumors.6 In contrast, COX-2 is not detected in the vasculature of normal tissues.27
Fig 2a. — COX-2 is expressed throughout mammary carcinogenesis. As
shown here, COX-2 is not detected in normal mammary epithelial cells
(normal) but is clearly expressed in ductal carcinoma in situ (DCIS),
ductal carcinoma, and infiltrating carcinoma. Formalin-fixed, paraffinembedded human cancers from archival tissues were prepared by
immunohistology using standard methods and stained for COX-2 with
isoform-specific antisera. The red stain depicts the presence of COX-2.
A
Normal
Primary CA
Hyperplasia
Metastasis to Lymph Node
B
Fig 2b. — COX-2 is up-regulated in colonic tumorigenesis. COX-2 is not
detected in normal cells. However, enhanced levels of COX-2 are observed in early colonic hyperplasia (sporadic adenomatous carcinoma),
primary colon cancer, and metastaticdisease. Formalin-fixed, paraffinembedded human cancers from archival tissues were prepared by
immunohistology using standard methods and stained for COX-2 with
isoform-specific antisera. The red stain depicts the presence of COX-2.
30 Cancer Control
C
Fig 3. — COX-2 immunoreactivity in human bladder cancer. Moderate-tostrong COX-2 immunoreactivity is detected in (a) the neoplastic epithelium (CA), (b) inflammatory (macrophage) cells, and (c) vasculature
endothelial cells (EC) within and adjacent to tumor nests. Tissue was
excised from patients with bladder cancer and stained for COX-2 with isoform-specific antisera. The red stain depicts the presence of COX-2.
March/April 2002, Vol. 9, No.2 Supplement
Our expression studies are consistent with those
reported by others28-38 and collectively imply COX-2
activity may be responsible for increased prostaglandin
levels in cancer tissues.39,40 Importantly, recent work
demonstrates a relationship between overexpression
of COX-2 and the invasiveness and survival of patients
with breast,41 colon,42-44 gastric,45,46 and lung47 cancers.
Association Between HER-2/neu and
COX-2 Expression
HER-2/neu, also known as c-erbB-2, is a receptor protein kinase whose overexpression is widely accepted as
an adverse prognostic marker.48 Interestingly, COX-2 is
detected in a subset of hormonally driven tumors such as
breast, prostate, and ovarian cancers.6 For example, COX2 overexpression in mammary tumors appears to be correlated with HER-2/neu. In a landmark study, Subbaramaiah et al49 evaluated COX-2 expression in 29 patients with
breast cancer and reported high levels of COX-2 in 14 of
15 HER-2/neu positive patients. In contrast, only 4 of 14
HER-2/neu negative patients expressed COX-2, and
detected levels were much lower than in any of the HER2/neu positive patients. Recently we observed that COX2 and HER-2/neu are similarly distributed in breast cancer
tissue and are often coexpressed in hyperproliferating,
dysplastic, and neoplastic epithelial cells.50 In another
study, we evaluated archival sections of human mammary
lesions, including DCIS, ductal, lobular, infiltrating, and
mucinous mammary cancers for HER-2/neu and COX-2.
Of the 25 cases evaluated, 15 were HER-2/neu positive
and 10 were HER-2/neu negative. Both COX-2 and HER2/neu were overexpressed in 3 of 3 cases of DCIS and 10
of 12 cases with ductal, lobular, and infiltrating carcinomas, and both were over expressed in the same cells.
COX-2 was expressed less often in HER-2/neu negative
breast cancers.51
To determine if these two oncogenes were functionally linked, HER-2/neu and COX-2 negative breast
cancer cells were engineered to express HER-2/neu.
COX-2 was strongly induced in HER-2/neu transfected
cells but was not expressed in null transfected cells.52
Collectively, these data suggest that HER-2/neu may
modulate the overexpression of COX-2 in human cancer.52 However, the upstream modulator(s) of COX-2 in
ovarian and prostate cancer have yet to be elucidated.
The correlation between COX-2 and HER-2/neu
expression in human breast cancers may have important
treatment implications. For example, patients with early
disease such as DCIS are often given the option of treatment vs no treatment after lumpectomy. These data collectively imply HER-2neu/COX-2 positive patients with
DCIS may represent a high-risk patient population that
March/April 2002, Vol. 9, No.2 Supplement
may benefit from treatment with celecoxib. Additionally,
combination therapy with celecoxib and other moleculartargeted agents such as aromatase inhibitors (ie, exemestane) or agents that block HER-2/neu activation may also
prove beneficial in clinical trials of breast cancer. Indeed,
future research on the effect of COX-2 inhibition on HER2/neu positive vs HER-2/neu negative mammary tumors
will significantly contribute to the present understanding
of the potential clinical utility of COX-2 inhibitors for the
prevention and/or treatment of human breast cancer.
Mechanisms of COX-2–Associated
Tumorigenesis
COX-2 is overexpressed along the continuum of
oncogenesis and is likely to be a key player in a number
of biologic pathways leading to cancer. Current evidence
indicates that COX-2 promotes tumor-specific angiogenesis,53-56 inhibits apoptosis,57-60 and induces proangiogenic
factors such as VEGF,61,62 inducible nitrogen oxide synthetase promoter (iNOS),63 IL-6,64 IL-8,65 and TIE-2.66
COX-2-derived metabolites from infiltrating inflammatory cells undoubtedly contribute to the tumorigenic process as well. For example, enhanced
prostaglandin synthesis may contribute to oncogenesis
by directly stimulating mitogenesis in fibroblasts,67
osteoblasts,68,69 and mammary epithelial cells.70 Excessive local synthesis of prostaglandins has also been
shown to disrupt immune surveillance that may otherwise suppress tumor growth.71,72 In addition, the direct
product of COX-2, PGH2, can isomerize by both enzymatic and nonenzymatic reactions to form the potent
mutagen malondialdehyde, which can induce frame
shifts and base pair substitutions.73 Additional free radical damage may occur via the peroxidative activity of
COX-2, which can efficiently oxidize aromatic and heterocyclic amines and dihydrodiol derivatives.74
Increased prostaglandin levels may be particularly
important during the progression of breast cancer. PGE2
has recently been shown to stimulate aromatase transcription,75,76 leading to supraphysiologic local estrogen
levels, which in turn leads to the subsequent release of
growth factors and enhanced proliferation.77 Immunohistochemical analysis by Brueggemeier et al78 supports
the association between COX-2 and aromatase. The
cytochrome P450 enzyme aromatase (CYP19) and COX2 were coexpressed in all cases of human breast cancer,
and a significant linear association was found between
levels of CYP19 gene expression and COX-2 gene
expression (R - 0.80, P<.0001).78
In addition to increasing aromatase transcription,
COX-2-induced PGE2 also promotes angiogenesis,53-55
Cancer Control 31
Fig 4. — Hypothetical mechanism by which celecoxib may enhance the
anticancer activity of aromatase inhibitors. COX-2-derived PGE2 potentiates estrogen biosynthesis by the cytochrome P450 enzyme aromatase and
promotes angiogenesis, tumor maintenance/growth, and metastasis. The
simultaneous inhibition of aromatase and COX-2 activity may therefore
enhance the anticancer activity of either agent alone.
which is required for tumor growth and metastasis.79,80
Taken together, these data suggest that both autocrine
and paracrine mechanisms may be responsible for the
development and/or progression of estrogen-dependent breast cancer either directly by stimulation of
tumor cell proliferation or indirectly by enhanced
prostaglandin-regulated local estrogen synthesis (Fig 4).
Anticancer Activity of COX Inhibitors
in Relevant Animal Models of Cancer
Retrospective and/or epidemiological studies of
conventional NSAID use and incidence of cancer led to
the hypothesis that COX-derived metabolites play an
important role in tumorigenesis. The inhibitory effect
of COX inhibitors on tumor growth in relevant animal
models of various epithelial cancers supports this
hypothesis.81-86 However, maximum efficacy in these
studies is typically dose-limited by COX-1-related toxicities. In contrast, COX-2 inhibitors have been shown to
markedly inhibit tumor growth and metastasis in several animal models of colon,87-90 skin,91,92 lung,6 bladder,93
and breast cancers.94,95
For example, sporadic colorectal adenomas and
adenocarcinomas can be generated by administering
azoxymethane (AOM). When tested in this model, celecoxib effectively prevents the development of colon
tumors in 93% of rats and inhibits tumor initiation by
approximately 80%.88,96 In this model, the chemopreventive effect of celecoxib was dose-related and treatment was effective during both initiation and postinitiation phases of the disease.87
Likewise, Harris et al94 evaluated the chemopreventive potential of celecoxib and ibuprofen in the
7,12-dimethyl-benz(a)anthracene (DMBA) model of
32 Cancer Control
breast cancer in female Sprague-Dawley rats and compared the results with untreated control animals. All
rats were pretreated 7 days before DMBA administration and therapy was continued for 105 days. Malignant tumors developed in 100% of control animals;
95% had multiple tumors and tumor size was >1.5 cm3.
Both celecoxib and ibuprofen significantly suppressed
the incidence, burden, and volume of malignant tumors
and prolonged the latency period of tumor induction
compared with controls. However, the effects of celecoxib were significantly more pronounced than those
of ibuprofen (Table).94 In a follow-up chemoprevention study in this model, Abou-Issa et al97 reported that
mammary tumor incidence, burden, and volume were
inhibited in a dose-dependent manner. Tumor incidence was 100% in controls vs 55%, 45%, and 25% in
rats fed a diet of celecoxib 500, 1,000, or 1,500 ppm,
respectively (P=.001).97
The chemotherapeutic activity of celecoxib was
next evaluated on established DMBA-induced mammary tumors by Alshafie and coworkers.95 DMBA-treated
rats were randomized to control or celecoxib following
6 weeks of DMBA treatment. Mammary tumors continued to grow in the control group, with an average
increase in tumor size of 500% vs baseline. In the celecoxib-treated group, tumor regression was reported in
approximately 90% of animals, and tumor was reduced
32% compared with baseline (P<.04). In contrast, 10
new mammary tumors were identified in the control
group (30% increase). The number of palpable tumors
decreased in celecoxib-treated animals from 24 to 18
(25%; P<.05); no decrease in the number of tumors was
observed in the control group.95
To identify the potential points of intervention during tumorigenesis, Alshafie et al98 dosed DMBA-treated
rats with celecoxib only during the initiation or promotion phases of carcinogenesis or during both initiation
and promotion phases. All treatment paradigms signifiChemopreventive Effects of Celecoxib and Ibuprofen:
Mammary Cancer Incidence, Tumor Burden, Tumor Volume, and
Latency of Tumor Induction in a DMBA Model of Breast Cancer
Effect vs Control
Celecoxib
Ibuprofen
Reduction in tumor incidence, %
68 *
†
40*
Reduction in tumor burden, %
86 *†
52*
Reduction in tumor volume, %
81 *
†
57*
Latency period of tumor induction, days‡
95
86
* P<.001 vs controls
† P<.01 vs ibuprofen
‡ 58 days in controls
Data from Harris et al.94
March/April 2002, Vol. 9, No.2 Supplement
cantly reduced tumor incidence and growth (P=.001),
with the effect being maximal when celecoxib was fed
during both the initiation and promotion phases.98
Based on the association between COX-2 and aromatase gene expression,75,78 Pesenti and colleagues99
evaluated the therapeutic potential of celecoxib (500
ppm in diet) and exemestane (50 mg/kg weekly) alone
and in combination in the DMBA model of breast cancer. Female Sprague-Dawley rats with at least one
tumor measuring 1 cm in size were studied. An objective response (OR) rate of 48% was achieved with combination therapy, compared with OR rates of 5% in rats
treated with exemestane alone and 0 in rats treated
with celecoxib alone and in control rats. The development of new tumors followed a similar pattern. These
data demonstrate that the combination of exemestane
and celecoxib is more effective than either agent alone
in reducing tumor burden and volume, as well as the
incidence of new tumors in this animal model.99 Moreover, it demonstrates that, while celecoxib inhibits the
exaggerated induction of aromatase, it does not eliminate baseline or physiologic aromatase production. The
addition of exemestane is required, therefore, to block
the activity of aromatase enzyme whose production is
not blocked by celecoxib.
Antiangiogenic agents have also been shown to
enhance the antitumor activity of cytotoxic drugs
when given in combination in rodent models of cancer.100,101 In addition to anti-inflammatory properties,
recent work convincingly demonstrates that celecoxib
can also act as a potent antiangiogenic agent.54 We
therefore evaluated the antitumor activity of celecoxib
as a monotherapy and in combination with cytotoxic
chemotherapy in the HT-29 human colon carcinoma
xenograft model and in the Lewis lung carcinoma syngeneic model. The combined modality of celecoxib
with cytotoxic agents was more effective in both cancer models than either agent alone. Recent work also
suggests that COX-2 inhibitors may potentiate radiation
therapy by increasing cellular radiosensitivity and greatly enhancing tumor response when these two modalities are administered in combination.102,103
Discussion and Conclusions
We have presented data to support the hypothesis
that COX-2 activity modulates critical steps in the initiation, promotion, and progression of several human
epithelial cancers. COX-2-derived prostaglandins may
promote the development of cancer by various mechanisms, including stimulation of tumor cell growth and
neovascularization.53-60,75,78,104-106 Thus, it is not surprising
that COX-2 inhibitors such as celecoxib markedly inhibit
March/April 2002, Vol. 9, No.2 Supplement
tumor growth and metastasis in a dose-dependent manner in numerous animal models of solid tumors.87,90-94
However, the exact manner by which celecoxib reduces
tumorigenesis has not been fully elucidated.
The ultimate goal of cancer treatment is to specifically prevent the growth of precancerous or cancerous
cells without affecting normal cells. This is particularly
important in chemoprevention and treatment of early
disease, which typically involves long-term treatment to
healthy subjects. Therefore, NSAID selection should be
based on safety as well as efficacy. Recent research has
clearly established that specific COX-2 inhibitors are
associated with less toxicity than the conventional
mixed COX inhibitors.10
In summary, COX-2 is overexpressed in both early
and late stages of carcinogenesis and has been shown to
be efficacious as monotherapy and in combination with
conventional chemotherapeutics in relevant animal
models. Taken together, the epidemiological data and
preclinical studies in animal models have generated
compelling interest in the potential use of COX-2
inhibitors in chemoprevention and chemotherapy of
human tumors. Clinical trials will be necessary to determine whether COX-2 inhibitors will provide clinical benefit, as well as to define the intervention points during
tumor progression that will allow for optimal efficacy.
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